1. Field of the Invention
The present invention relates to a microorganism belonging to the family Enterobacteriaceae including L-carnitine biosynthesis-associated genes derived from Neurospora crassa, and a process for producing L-carnitine using the same.
2. Description of the Related Art
L-carnitine (3-hydroxy-4-trimethylaminobutyrate), which is commonly present in an organism, is a zwitterionic compound responsible for the transportation of activated long-chain fatty acids into the mitochondrial matrix via the membrane of the mitochondria. It is known that L-carnitine is biosynthesized from lysine or lysine in protein (hereinafter, referred to as “protein lysine”). Mammalian protein lysine is generally used as a precursor for L-carnitine biosynthesis. However, in Neurospora crassa, free lysine is used as a precursor of L-carnitine. In the biosynthesis of L-carnitine, ε-N,N,N-trimethyllysine, ε-N,N,N-trimethyl-β-hydroxylysine, N,N-trimethylaminobutyraldehyde, and γ-butyrobetaine are formed as intermediates. γ-butyrobetaine is hydroxylated to L-carnitine by γ-butyrobetaine hydroxylase. FIG. 1 illustrates putative L-carnitine biosynthesis pathway in Neurospora crassa. 
L-carnitine can be produced by chemical synthesis, enzymatic semisynthesis, or microbiological method. However, the chemical synthesis of carnitine unavoidably leads to DL-carnitine racemic mixtures, and thus requires separation of the DL-racemic mixtures. With respect to the enzymatic semisynthesis of L-carnitine, for example, U.S. Pat. No. 4,221,869 discloses a process for producing L-carnitine from dehydrocarnitine using carnitine dehydrogenase (EC 1.1.1.108) and a coenzyme, NAD. However, dehydrocarnitine is very unstable and thus can be spontaneously decomposed into acetonyltrimethylammonium and carbon dioxide. German Patent No. DE-OS-3123975 discloses a process for producing L-carnitine from γ-butyrobetaine using γ-butyrobetaine hydroxylase (EC 1.14.11.1) isolated from Neurospora crassa. However, there is a disadvantage that α-ketoglutarate and a reducing agent (i.e., ascorbate) must be added to the reaction mixture during hydroxylation.
With respect to the production of L-carnitine by microbiological method, U.S. Pat. No. 5,028,538 discloses a process for producing L-carnitine, which includes incubating E. coli 044 K 74 in a culture medium containing crotonobetaine (4-N,N,N-triethylaminocrotonic acid) and recovering L-carnitine from the culture. U.S. Pat. No. 4,708,936 discloses a process for producing L-carnitine by incubating Achromobacter xylosoxydans DSM 3225 (HK 1331b) in a crotonobetaine- and/or γ-butyrobetaine-containing medium. According to this process, however, the use of crotonobetaine which is neither a precursor nor an intermediate for L-carnitine biosynthesis is required and the yield of L-carnitine is not high. Thus, the microbiological method needs to improve the yield of L-carnitine.
Therefore, while searching for L-carnitine-producing microorganism capable of producing L-carnitine with high yield using an inexpensive precursor, the present inventors found that L-carnitine biosynthesis-associated genes derived from Neurospora crassa were well expressed in a microorganism belonging to the family Enterobacteriaceae, and thus completed the present invention.